1. Field of the Invention
A significant number of cellular functions are predicated on the ability to secrete certain polypeptides. The signal for secretion appears to be a leader sequence which is predominantly hydrophobic in nature joined to the mature peptide through a processing oligopeptide signal recognized by an endopeptidase. The ability to secrete polypeptides foreign to a host is very attractive, since there may be substantially less degradation of the desired product and where the natural product has an amino acid other than methionine as the first amino acid, the secreted product will have the correct N-terminus. Where secretion is obtained at high efficiency a more efficient and economic production of the desired polypeptide may result.
Secretion of the desired polypeptide has many attractions. As indicated, the probability of degradation of the polypeptide is diminished, where the intracellular lifetime of the polypeptide is short. Furthermore, where the polypeptide is retained intracellularly, it is necessary to isolate and lyse the cells, and separate the polypeptide of interest from the total cellular protein. By contrast, where the polypeptide is secreted, one can continuously withdraw the nutrient medium, isolating the protein of interest where the protein background is substantially smaller. Furthermore, in many cases the enhanced concentration of the polypeptide in the host can be lethal to the host or substantially diminish the viability of the host. In these cases, the production of the deslies polypeptide will be relatively small. Where a fused polypeptide is employed, having a signal leader sequence, the signal leader may not only serve to provide for secretion, but also mask the lethal character of the foreign polypeptide.
There are a substantial number of different endopeptidases present in cells. These endopeptidases have different specificities, so as to require different sequences and/or conformations of the polypeptide as the cleavage site. With hybrid DNA technology, one tries to provide a high level of production of a polypeptide product, which is in addition to the normal cellular products. Where such polypeptide requires processing, the cell may not be able to respond to the increased processing load. However, the mere fact of providing for enhanced genetic capability of producing the endopeptidase is no assurance that there will be an enhanced or more efficient processing of the endopeptidase substrate.
2. Description of the Prior Art
S. cerevisiae mutants having a lesion associated with killer toxin expression and .alpha.-factor pheromone expression are described by Wickner and Leibowitz, Genetics (1976) 82:429-442; Leibowitz and Wickner, Proc. Natl. Acad. Sci. USA (1976) 73:2061-2065. The required processing of killer toxin and .alpha.-factor precursors is described by Bostian et al., Cell (1983) 32:169-180; Bussey et al., Molec. Cellul. Biol. (1983) 3:1362-1370 and Skipper et al., EMBO J. (1984) 3:107-111, Julius et al., Cell (1983) 32:839-852 and Julius et al., Cell (1984) 36:309-318. Mapping of the lesion associated with loss of killer activity may be found in Wickner and Leibowitz (1976), supra; and Mortimer and Schild, (1982) "Genetic Map of Saccharomyces cerevisiae." In Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, (Strathern, J. N., Jones, E. W., and Broach, J. R., Eds.) Cold Spring Harbor Laboratory, NY, pp. 639-350. Proteinase M reported by Achstetter et al., Arch. Biochem. Biophys. (1981) 207:445-454, appears to have activities analogous to the product of the KEX2 gene.